Degradomic Identification of Membrane Type 1-Matrix Metalloproteinase as an ADAMTS9 and ADAMTS20 Substrate.

The secreted metalloproteases ADAMTS9 and ADAMTS20 are implicated in extracellular matrix proteolysis and primary cilium biogenesis. Here, we show that clonal gene-edited RPE-1 cells in which ADAMTS9 was inactivated, and which constitutively lack ADAMTS20 expression, have morphologic characteristics distinct from parental RPE-1 cells. To investigate underlying proteolytic mechanisms, a quantitative terminomics method, terminal amine isotopic labeling of substrates was used to compare the parental and gene-edited RPE-1 cells and their medium to identify ADAMTS9 substrates. Among differentially abundant neo-amino (N) terminal peptides arising from secreted and transmembrane proteins, a peptide with lower abundance in the medium of gene-edited cells suggested cleavage at the Tyr314-Gly315 bond in the ectodomain of the transmembrane metalloprotease membrane type 1-matrix metalloproteinase (MT1-MMP), whose mRNA was also reduced in gene-edited cells. This cleavage, occurring in the MT1-MMP hinge, that is, between the catalytic and hemopexin domains, was orthogonally validated both by lack of an MT1-MMP catalytic domain fragment in the medium of gene-edited cells and restoration of its release from the cell surface by reexpression of ADAMTS9 and ADAMTS20 and was dependent on hinge O-glycosylation. A C-terminally semitryptic MT1-MMP peptide with greater abundance in WT RPE-1 medium identified a second ADAMTS9 cleavage site in the MT1-MMP hemopexin domain. Consistent with greater retention of MT1-MMP on the surface of gene-edited cells, pro-MMP2 activation, which requires cell surface MT1-MMP, was increased. MT1-MMP knockdown in gene-edited ADAMTS9/20-deficient cells restored focal adhesions but not ciliogenesis. The findings expand the web of interacting proteases at the cell surface, suggest a role for ADAMTS9 and ADAMTS20 in regulating cell surface activity of MT1-MMP, and indicate that MT1-MMP shedding does not underlie their observed requirement in ciliogenesis.

Emerging roles for the ADAMTS-like family of matricellular proteins in cardiovascular disease through regulation of the extracellular microenvironment.

Dysregulation of the extracellular matrix (ECM) occurs widely across cardiovascular pathologies. Recent work has revealed important roles for the «a disintegrin-like and metalloprotease domain with thrombospondin-type 1 motifs like" (ADAMTSL) family of secreted glycoproteins in cardiovascular tissues during development and disease. Key insights in this regard have come from naturally occurring gene mutations in humans and animals that result in severe diseases with cardiovascular manifestations or aortopathies. Expression of ADAMTSL genes is greatly increased in the myocardium during heart failure. Genetically modified mice recapitulate phenotypes of patients with ADAMTSL mutations and demonstrate important functions in the ECM. The novel functions thus disclosed are intriguing because, while these proteins are neither structural, nor proteases like the related ADAMTS proteases, they appear to act as regulatory, i.e., matricellular proteins. Evidence from genetic variants, genetically engineered mouse mutants, and in vitro investigations have revealed regulatory functions of ADAMTSLs related to fibrillin microfibrils and growth factor signaling. Interestingly, the ability to regulate transforming growth factor (TGF)ß signaling may be a shared characteristic of some ADAMTSLs. TGFß signaling is important in cardiovascular development, health and disease and a central driver of ECM remodeling and cardiac fibrosis. New strategies to target dysregulated TGFß signaling are warranted in aortopathies and cardiac fibrosis. With their emerging roles in cardiovascular tissues, the ADAMTSL proteins may provide causative genes, diagnostic biomarkers and novel treatment targets in cardiovascular disease. Here, we discuss the relevance of ADAMTSLs to cardiovascular medicine.

Regulation of extracellular matrix composition by fibroblasts during perinatal cardiac maturation.

BACKGROUND: Cardiac fibroblasts are the main non-myocyte population responsible for extracellular matrix (ECM) production. During perinatal development, fibroblast expansion coincides with the transition from hyperplastic to hypertrophic myocardial growth. Therefore, we investigated the consequences of fibroblast loss at the time of cardiomyocyte maturation by depleting fibroblasts in the perinatal mouse. METHODS AND RESULTS: We evaluated the microenvironment of the perinatal heart in the absence of fibroblasts and the potential functional impact of fibroblast loss in regulation of cardiomyocyte cell cycle arrest and binucleation. Cre-mediated expression of diphtheria toxin A in PDGFRα expressing cells immediately after birth eliminated 70-80% of the cardiac fibroblasts. At postnatal day 5, hearts lacking fibroblasts appeared similar to controls with normal morphology and comparable numbers of endothelial and smooth muscle cells, despite a pronounced reduction in fibrillar collagen. Immunoblotting and proteomic analysis of control and fibroblast-deficient hearts identified differential abundance of several ECM proteins. In addition, fibroblast loss decreased tissue stiffness and resulted in increased cardiomyocyte mitotic index, DNA synthesis, and cytokinesis. Moreover, decellularized matrix from fibroblast-deficient hearts promoted cardiomyocyte DNA replication. While cardiac architecture was not overtly affected by fibroblast reduction, few pups survived past postnatal day 11, suggesting an overall requirement for PDGFRα expressing fibroblasts. CONCLUSIONS: These studies demonstrate the key role of fibroblasts in matrix production and cardiomyocyte cross-talk during mouse perinatal heart maturation and revealed that fibroblast-derived ECM may modulate cardiomyocyte maturation in vivo. Neonatal depletion of fibroblasts demonstrated that although hearts can tolerate reduced ECM composition, fibroblast loss eventually leads to perinatal death as the approach simultaneously reduced fibroblast populations in other organs.

Proteolysis: a key post-translational modification regulating proteoglycans.

Proteoglycans are composite molecules comprising a protein backbone, i.e., the core protein, with covalently attached glycosaminoglycan chains of distinct chemical types. Most proteoglycans are secreted or attached to the cell membrane. Their specialized structures, binding properties, and biophysical attributes underlie diverse biological roles, which include modulation of tissue mechanics, cell adhesion, and the sequestration and regulated release of morphogens, growth factors, and cytokines. As an irreversible post-translational modification, proteolysis has a profound impact on proteoglycan function, abundance, and localization. Proteolysis is required for molecular maturation of some proteoglycans, clearance of extracellular matrix proteoglycans during tissue remodeling, generation of bioactive fragments from proteoglycans, and ectodomain shedding of cell-surface proteoglycans. Genetic evidence shows that proteoglycan core protein proteolysis is essential for diverse morphogenetic events during embryonic development. In contrast, dysregulated proteoglycan proteolysis contributes to osteoarthritis, cardiovascular disorders, cancer, and inflammation. Proteolytic fragments of perlecan, versican, aggrecan, brevican, collagen XVIII, and other proteoglycans are associated with independent biological activities as so-called matrikines. Yet, proteoglycan proteolysis has been investigated to only a limited extent to date. Here, we review the actions of proteases on proteoglycans and illustrate their functional impact with several examples. We discuss the applications and limitations of strategies used to define cleavage sites in proteoglycans and explain how proteoglycanome-wide proteolytic mapping, which is desirable to fully understand the impact of proteolysis on proteoglycans, can be facilitated by integrating classical proteoglycan isolation methods with mass spectrometry-based proteomics.

Post-translational regulation and proteolytic activity of the metalloproteinase ADAMTS8.

A disintegrin-like and metalloprotease domain with thrombospondin type 1 motifs (ADAMTS)8 is a secreted protease, which was recently implicated in pathogenesis of pulmonary arterial hypertension (PAH). However, the substrate repertoire of ADAMTS8 and regulation of its activity are incompletely understood. Although considered a proteoglycanase because of high sequence similarity and close phylogenetic relationship to the proteoglycan-degrading proteases ADAMTS1, 4, 5, and 15, as well as tight genetic linkage with ADAMTS15 on human chromosome 11, its aggrecanase activity was reportedly weak. Several post-translational factors are known to regulate ADAMTS proteases such as autolysis, inhibition by endogenous inhibitors, and receptor-mediated endocytosis, but their impacts on ADAMTS8 are unknown. Here, we show that ADAMTS8 undergoes autolysis at six different sites within its spacer domain. We also found that in contrast to ADAMTS4 and 5, ADAMTS8 levels were not regulated through low-density lipoprotein receptor-related protein 1 (LRP1)-mediated endocytosis. Additionally, ADAMTS8 lacked significant activity against the proteoglycans aggrecan, versican, and biglycan. Instead, we found that ADAMTS8 cleaved osteopontin, a phosphoprotein whose expression is upregulated in PAH. Multiple ADAMTS8 cleavage sites were identified using liquid chromatography-tandem mass spectrometry. Osteopontin cleavage by ADAMTS8 was efficiently inhibited by TIMP-3, an endogenous inhibitor of ADAMTS1, 4, and 5, as well as by TIMP-2, which has no previously reported inhibitory activity against other ADAMTS proteases. These differences in post-translational regulation and substrate repertoire differentiate ADAMTS8 from other family members and may help to elucidate its role in PAH.

Mutations of ADAMTS9 Cause Nephronophthisis-Related Ciliopathy.

Nephronophthisis-related ciliopathies (NPHP-RCs) are a group of inherited diseases that are associated with defects in primary cilium structure and function. To identify genes mutated in NPHP-RC, we performed homozygosity mapping and whole-exome sequencing for >100 individuals, some of whom were single affected individuals born to consanguineous parents and some of whom were siblings of indexes who were also affected by NPHP-RC. We then performed high-throughput exon sequencing in a worldwide cohort of 800 additional families affected by NPHP-RC. We identified two ADAMTS9 mutations (c.4575_4576del [p.Gln1525Hisfs∗60] and c.194C>G [p.Thr65Arg]) that appear to cause NPHP-RC. Although ADAMTS9 is known to be a secreted extracellular metalloproteinase, we found that ADAMTS9 localized near the basal bodies of primary cilia in the cytoplasm. Heterologously expressed wild-type ADAMTS9, in contrast to mutant proteins detected in individuals with NPHP-RC, localized to the vicinity of the basal body. Loss of ADAMTS9 resulted in shortened cilia and defective sonic hedgehog signaling. Knockout of Adamts9 in IMCD3 cells, followed by spheroid induction, resulted in defective lumen formation, which was rescued by an overexpression of wild-type, but not of mutant, ADAMTS9. Knockdown of adamts9 in zebrafish recapitulated NPHP-RC phenotypes, including renal cysts and hydrocephalus. These findings suggest that the identified mutations in ADAMTS9 cause NPHP-RC and that ADAMTS9 is required for the formation and function of primary cilia.

Alternative splicing of the metalloprotease ADAMTS17 spacer regulates secretion and modulates autoproteolytic activity.

ADAMTS proteases mediate biosynthesis and breakdown of secreted extracellular matrix (ECM) molecules in numerous physiological and disease processes. In addition to their catalytic domains, ADAMTS proteases contain ancillary domains, which mediate substrate recognition and ECM binding and confer distinctive properties and roles to individual ADAMTS proteases. Although alternative splicing can greatly expand the structural and functional diversity of ADAMTS proteases, it has been infrequently reported and functional consequences have been rarely investigated. Here, we characterize the structural and functional impact of alternative splicing of ADAMTS17, mutations in which cause Weill-Marchesani syndrome 4. Two novel ADAMTS17 splice variants, ADAMTS17A and ADAMTS17B, were investigated by structural modeling, mass spectrometry, and biochemical approaches. Our results identify a novel disulfide-bridged insertion in the ADAMTS17A spacer that originates from inclusion of a novel exon. This insertion results in differential autoproteolysis of ADAMTS17, and thus, predicts altered proteolytic activity against other substrates. The second variant, ADAMTS17B, results from an in-frame exon deletion and prevents ADAMTS17B secretion. Thus, alternative splicing of the ADAMTS spacer significantly regulates the physiologically relevant proteolytic activity of ADAMTS17, either by altering proteolytic specificity (ADAMTS17A) or by altering cellular localization (ADAMTS17B).

O-Fucosylation of ADAMTSL2 is required for secretion and is impacted by geleophysic dysplasia-causing mutations.

ADAMTSL2 mutations cause an autosomal recessive connective tissue disorder, geleophysic dysplasia 1 (GPHYSD1), which is characterized by short stature, small hands and feet, and cardiac defects. ADAMTSL2 is a matricellular protein previously shown to interact with latent transforming growth factor-ß binding protein 1 and influence assembly of fibrillin 1 microfibrils. ADAMTSL2 contains seven thrombospondin type-1 repeats (TSRs), six of which contain the consensus sequence for O-fucosylation by protein O-fucosyltransferase 2 (POFUT2). O-fucose-modified TSRs are subsequently elongated to a glucose ß1-3-fucose (GlcFuc) disaccharide by ß1,3-glucosyltransferase (B3GLCT). B3GLCT mutations cause Peters Plus Syndrome (PTRPLS), which is characterized by skeletal defects similar to GPHYSD1. Several ADAMTSL2 TSRs also have consensus sequences for C-mannosylation. Six reported GPHYSD1 mutations occur within the TSRs and two lie near O-fucosylation sites. To investigate the effects of TSR glycosylation on ADAMTSL2 function, we used MS to identify glycan modifications at predicted consensus sequences on mouse ADAMTSL2. We found that most TSRs were modified with the GlcFuc disaccharide at high stoichiometry at O-fucosylation sites and variable mannose stoichiometry at C-mannosylation sites. Loss of ADAMTSL2 secretion in POFUT2-/- but not in B3GLCT-/- cells suggested that impaired ADAMTSL2 secretion is not responsible for skeletal defects in PTRPLS patients. In contrast, secretion was significantly reduced for ADAMTSL2 carrying GPHYSD1 mutations (S641L in TSR3 and G817R in TSR6), and S641L eliminated O-fucosylation of TSR3. These results provide evidence that abnormalities in GPHYSD1 patients with this mutation are caused by loss of O-fucosylation on TSR3 and impaired ADAMTSL2 secretion.

ADAMTS9 and ADAMTS20 are differentially affected by loss of B3GLCT in mouse model of Peters plus syndrome.

Peters plus syndrome (MIM #261540 PTRPLS), characterized by defects in eye development, prominent forehead, hypertelorism, short stature and brachydactyly, is caused by mutations in the ß3-glucosyltransferase (B3GLCT) gene. Protein O-fucosyltransferase 2 (POFUT2) and B3GLCT work sequentially to add an O-linked glucose ß1-3fucose disaccharide to properly folded thrombospondin type 1 repeats (TSRs). Forty-nine proteins are predicted to be modified by POFUT2, and nearly half are members of the ADAMTS superfamily. Previous studies suggested that O-linked fucose is essential for folding and secretion of POFUT2-modified proteins and that B3GLCT-mediated extension to the disaccharide is essential for only a subset of targets. To test this hypothesis and gain insight into the origin of PTRPLS developmental defects, we developed and characterized two mouse B3glct knockout alleles. Using these models, we tested the role of B3GLCT in enabling function of ADAMTS9 and ADAMTS20, two highly conserved targets whose functions are well characterized in mouse development. The mouse B3glct mutants developed craniofacial and skeletal abnormalities comparable to PTRPLS. In addition, we observed highly penetrant hydrocephalus, white spotting and soft tissue syndactyly. We provide strong genetic and biochemical evidence that hydrocephalus and white spotting in B3glct mutants resulted from loss of ADAMTS20, eye abnormalities from partial reduction of ADAMTS9 and cleft palate from loss of ADAMTS20 and partially reduced ADAMTS9 function. Combined, these results provide compelling evidence that ADAMTS9 and ADAMTS20 were differentially sensitive to B3GLCT inactivation and suggest that the developmental defects in PTRPLS result from disruption of a subset of highly sensitive POFUT2/B3GLCT targets such as ADAMTS20.

ADAMTSL5 is an epigenetically activated gene underlying tumorigenesis and drug resistance in hepatocellular carcinoma.

BACKGROUND & AIMS: The tumour microenvironment shapes tumour growth through cellular communications that include both direct interactions and secreted factors. The aim of this study was to characterize the impact of the secreted glycoprotein ADAMTSL5, whose role in cancer has not been previously investigated, on hepatocellular carcinoma (HCC). METHODS: ADAMTSL5 methylation status was evaluated through bisulfite sequencing, and publicly available data analysis. ADAMTSL5 RNA and protein expression were assessed in mouse models and HCC patient samples and compared to data from published datasets. Functional studies, including association of ADAMTSL5 depletion with responsiveness to clinically relevant drugs, were performed in cellular and in vivo models. Molecular alterations associated with ADAMTSL5 targeting were determined using proteomics, biochemistry, and reverse-transcription quantitative PCR. RESULTS: Methylome analysis revealed hypermethylated gene body CpG islands at the ADAMTSL5 locus in both mouse and human HCC, correlating with higher ADAMTSL5 expression. ADAMTSL5 targeting interfered with tumorigenic properties of HCC cells in vitro and in vivo, whereas ADAMTSL5 overexpression conferred tumorigenicity to pre-tumoural hepatocytes sensitized to transformation by a modest level of MET receptor expression. Mechanistically, ADAMTSL5 abrogation led to a reduction of several oncogenic inputs relevant to HCC, including reduced expression and/or phosphorylation levels of receptor tyrosine kinases MET, EGFR, PDGFRß, IGF1Rß, or FGFR4. This phenotype was associated with significantly increased sensitivity of HCC cells to clinically relevant drugs, namely sorafenib, lenvatinib, and regorafenib. Moreover, ADAMTSL5 depletion drastically increased expression of AXL, accompanied by a sensitization to bemcentinib. CONCLUSIONS: Our results point to a role for ADAMTSL5 in maintaining the function of key oncogenic signalling pathways, suggesting that it may act as a master regulator of tumorigenicity and drug resistance in HCC. LAY SUMMARY: The environment of cancer cells has profound effects on establishment, progression, and response of a tumour to treatment. Herein, we show that ADAMTSL5, a protein secreted by liver cancer cells and overlooked in cancer so far, is increased in this tumour type, is necessary for tumour formation and supports drug resistance. Adamtsl5 removal conferred sensitivity of liver cancer cells to drugs used in current treatment. This suggests ADAMTSL5 as a potential marker in liver cancer as well as a possible drug target.

A disintegrin-like and metalloproteinase domain with thrombospondin type 1 motif 9 (ADAMTS9) regulates fibronectin fibrillogenesis and turnover.

The secreted metalloprotease ADAMTS9 has dual roles in extracellular matrix (ECM) turnover and biogenesis of the primary cilium during mouse embryogenesis. Its gene locus is associated with several human traits and disorders, but ADAMTS9 has few known interacting partners or confirmed substrates. Here, using a yeast two-hybrid screen for proteins interacting with its C-terminal Gon1 domain, we identified three putative ADAMTS9-binding regions in the ECM glycoprotein fibronectin. Using solid-phase binding assays and surface plasmon resonance experiments with purified proteins, we demonstrate that ADAMTS9 and fibronectin interact. ADAMTS9 constructs, including those lacking Gon1, co-localized with fibronectin fibrils formed by cultured fibroblasts lacking fibrillin-1, which co-localizes with fibronectin and binds several ADAMTSs. We observed no fibrillar ADAMTS9 staining after blockade of fibroblast fibronectin fibrillogenesis with a peptide based on the functional upstream domain of a Staphylococcus aureus adhesin. These findings indicate that ADAMTS9 binds fibronectin dimers and fibrils directly through multiple sites in both molecules. Proteolytically active ADAMTS9, but not a catalytically inactive variant, disrupted fibronectin fibril networks formed by fibroblasts in vitro, and ADAMTS9-deficient RPE1 cells assembled a robust fibronectin fibril network, unlike WT cells. Targeted LC-MS analysis of fibronectin digested by ADAMTS9-expressing cells identified a semitryptic peptide arising from cleavage at Gly2196-Leu2197 We noted that this scissile bond is in the linker between fibronectin modules III17 and I10, a region targeted also by other proteases. These findings, along with stronger fibronectin staining previously observed in Adamts9 mutant embryos, suggest that ADAMTS9 contributes to fibronectin turnover during ECM remodeling.

Disruption of the Extracellular Matrix Progressively Impairs Central Nervous System Vascular Maturation Downstream of ß-Catenin Signaling.

Objective- The Wnt/ß-catenin pathway orchestrates development of the blood-brain barrier, but the downstream mechanisms involved at different developmental windows and in different central nervous system (CNS) tissues have remained elusive. Approach and Results- Here, we create a new mouse model allowing spatiotemporal investigations of Wnt/ß-catenin signaling by induced overexpression of Axin1, an inhibitor of ß-catenin signaling, specifically in endothelial cells ( Axin1 iEC- OE). AOE (Axin1 overexpression) in Axin1 iEC- OE mice at stages following the initial vascular invasion of the CNS did not impair angiogenesis but led to premature vascular regression followed by progressive dilation and inhibition of vascular maturation resulting in forebrain-specific hemorrhage 4 days post-AOE. Analysis of the temporal Wnt/ß-catenin driven CNS vascular development in zebrafish also suggested that Axin1 iEC- OE led to CNS vascular regression and impaired maturation but not inhibition of ongoing angiogenesis within the CNS. Transcriptomic profiling of isolated, ß-catenin signaling-deficient endothelial cells during early blood-brain barrier-development (E11.5) revealed ECM (extracellular matrix) proteins as one of the most severely deregulated clusters. Among the 20 genes constituting the forebrain endothelial cell-specific response signature, 8 ( Adamtsl2, Apod, Ctsw, Htra3, Pglyrp1, Spock2, Ttyh2, and Wfdc1) encoded bona fide ECM proteins. This specific ß-catenin-responsive ECM signature was also repressed in Axin1 iEC- OE and endothelial cell-specific ß-catenin-knockout mice ( Ctnnb1-KOiEC) during initial blood-brain barrier maturation (E14.5), consistent with an important role of Wnt/ß-catenin signaling in orchestrating the development of the forebrain vascular ECM. Conclusions- These results suggest a novel mechanism of establishing a CNS endothelium-specific ECM signature downstream of Wnt-ß-catenin that impact spatiotemporally on blood-brain barrier differentiation during forebrain vessel development. Visual Overview- An online visual overview is available for this article.

A Selective Extracellular Matrix Proteomics Approach Identifies Fibronectin Proteolysis by A Disintegrin-like and Metalloprotease Domain with Thrombospondin Type 1 Motifs (ADAMTS16) and Its Impact on Spheroid Morphogenesis.

Secreted and cell-surface proteases are major mediators of extracellular matrix (ECM) turnover, but their mechanisms and regulatory impact are poorly understood. We developed a mass spectrometry approach using a cell-free ECM produced in vitro to identify fibronectin (FN) as a novel substrate of the secreted metalloprotease ADAMTS16. ADAMTS16 cleaves FN between its (I)5 and (I)6 modules, releasing the N-terminal 30 kDa heparin-binding domain essential for FN self-assembly. ADAMTS16 impairs FN fibrillogenesis as well as fibrillin-1 and tenascin-C assembly, thus inhibiting formation of a mature ECM by cultured fibroblasts. Furthermore ADAMTS16 has a marked morphogenetic impact on spheroid formation by renal tubule-derived MDCKI cells. The N-terminal FN domain released by ADAMTS16 up-regulates MMP3, which cleaves the (I)5-(I)6 linker of FN similar to ADAMTS16, therefore creating a proteolytic feed-forward mechanism. Thus, FN proteolysis not only regulates FN turnover, but also FN assembly, with potential long-term consequences for ECM assembly and morphogenesis.

Immunoregulatory roles of versican proteolysis in the myeloma microenvironment.

Myeloma immunosurveillance remains incompletely understood. We have demonstrated proteolytic processing of the matrix proteoglycan, versican (VCAN), in myeloma tumors. Whereas intact VCAN exerts tolerogenic activities through Toll-like receptor 2 (TLR2) binding, the immunoregulatory consequences of VCAN proteolysis remain unknown. Here we show that human myeloma tumors displaying CD8(+) infiltration/aggregates underwent VCAN proteolysis at a site predicted to generate a glycosaminoglycan-bereft N-terminal fragment, versikine Myeloma-associated macrophages (MAMs), rather than tumor cells, chiefly produced V1-VCAN, the precursor to versikine, whereas stromal cell-derived ADAMTS1 was the most robustly expressed VCAN-degrading protease. Purified versikine induced early expression of inflammatory cytokines interleukin 1ß (IL-1ß) and IL-6 by human myeloma marrow-derived MAMs. We show that versikine signals through pathways both dependent and independent of Tpl2 kinase, a key regulator of nuclear factor κB1-mediated MAPK activation in macrophages. Unlike intact VCAN, versikine-induced Il-6 production was partially independent of Tlr2. In a model of macrophage-myeloma cell crosstalk, versikine induced components of "T-cell inflammation," including IRF8-dependent type I interferon transcriptional signatures and T-cell chemoattractant CCL2. Thus the interplay between stromal cells and myeloid cells in the myeloma microenvironment generates versikine, a novel bioactive damage-associated molecular pattern that may facilitate immune sensing of myeloma tumors and modulate the tolerogenic consequences of intact VCAN accumulation. Therapeutic versikine administration may potentiate T-cell-activating immunotherapies.

Identification and functional analysis of an ADAMTSL1 variant associated with a complex phenotype including congenital glaucoma, craniofacial, and other systemic features in a three-generation human pedigree.

Developmental glaucoma can occur as an isolated or syndromic condition and is genetically heterogeneous. We describe a three-generation family affected with developmental glaucoma, myopia, and/or retinal defects associated with variable craniofacial/dental, auditory, brain, renal, and limb anomalies. Whole-exome sequencing identified a heterozygous c.124T> C, p.(Trp42Arg) allele in ADAMTSL1; cosegregation analysis confirmed the presence of this allele in four affected family members. The mutation affects a highly conserved residue and is strongly predicted to have a deleterious effect on protein function. Trp42 is normally modified by protein C-mannosylation, an unusual post-translational modification. Comparison of ADAMTSL1-WT (also known as punctin-1) and ADAMTSL1-p.Trp42Arg in vitro demonstrated that the latter was not secreted from transfected cells but retained intracellularly. Moreover, ADAMTSL1-p.Trp42Arg reduced secretion of cotransfected wild-type ADAMTSL1, suggesting a dominant negative effect for this mutation. These data imply a multisystem role for ADAMTSL1 and present the first disease-associated variant affecting a C-mannosylation motif.

Genetic and biochemical evidence that gastrulation defects in Pofut2 mutants result from defects in ADAMTS9 secretion.

Protein O-fucosyltransferase 2 (POFUT2) adds O-linked fucose to Thrombospondin Type 1 Repeats (TSR) in 49 potential target proteins. Nearly half the POFUT2 targets belong to the A Disintegrin and Metalloprotease with ThromboSpondin type-1 motifs (ADAMTS) or ADAMTS-like family of proteins. Both the mouse Pofut2 RST434 gene trap allele and the Adamts9 knockout were reported to result in early embryonic lethality, suggesting that defects in Pofut2 mutant embryos could result from loss of O-fucosylation on ADAMTS9. To address this question, we compared the Pofut2 and Adamts9 knockout phenotypes and used Cre-mediated deletion of Pofut2 and Adamts9 to dissect the tissue-specific role of O-fucosylated ADAMTS9 during gastrulation. Disruption of Pofut2 using the knockout (LoxP) or gene trap (RST434) allele, as well as deletion of Adamts9, resulted in disorganized epithelia (epiblast, extraembryonic ectoderm, and visceral endoderm) and blocked mesoderm formation during gastrulation. The similarity between Pofut2 and Adamts9 mutants suggested that disruption of ADAMTS9 function could be responsible for the gastrulation defects observed in Pofut2 mutants. Consistent with this prediction, CRISPR/Cas9 knockout of POFUT2 in HEK293T cells blocked secretion of ADAMTS9. We determined that Adamts9 was dynamically expressed during mouse gastrulation by trophoblast giant cells, parietal endoderm, the most proximal visceral endoderm adjacent to the ectoplacental cone, extraembryonic mesoderm, and anterior primitive streak. Conditional deletion of either Pofut2 or Adamts9 in the epiblast rescues the gastrulation defects, and identified a new role for O-fucosylated ADAMTS9 during morphogenesis of the amnion and axial mesendoderm. Combined, these results suggested that loss of ADAMTS9 function in the extra embryonic tissue is responsible for gastrulation defects in the Pofut2 knockout. We hypothesize that loss of ADAMTS9 function in the most proximal visceral endoderm leads to slippage of the visceral endoderm and altered characteristics of the extraembryonic ectoderm. Consequently, loss of input from the extraembryonic ectoderm and/or compression of the epiblast by Reichert's membrane blocks gastrulation. In the future, the Pofut2 and Adamts9 knockouts will be valuable tools for understanding how local changes in the properties of the extracellular matrix influence the organization of tissues during mammalian development.

Disruption of murine Adamtsl4 results in zonular fiber detachment from the lens and in retinal pigment epithelium dedifferentiation.

Human gene mutations have revealed that a significant number of ADAMTS (a disintegrin-like and metalloproteinase (reprolysin type) with thrombospondin type 1 motifs) proteins are necessary for normal ocular development and eye function. Mutations in human ADAMTSL4, encoding an ADAMTS-like protein which has been implicated in fibrillin microfibril biogenesis, cause ectopia lentis (EL) and EL et pupillae. Here, we report the first ADAMTSL4 mouse model, tvrm267, bearing a nonsense mutation in Adamtsl4. Homozygous Adamtsl4(tvrm267) mice recapitulate the EL phenotype observed in humans, and our analysis strongly suggests that ADAMTSL4 is required for stable anchorage of zonule fibers to the lens capsule. Unexpectedly, homozygous Adamtsl4(tvrm267) mice exhibit focal retinal pigment epithelium (RPE) defects primarily in the inferior eye. RPE dedifferentiation was indicated by reduced pigmentation, altered cellular morphology and a reduction in RPE-specific transcripts. Finally, as with a subset of patients with ADAMTSL4 mutations, increased axial length, relative to age-matched controls, was observed and was associated with the severity of the RPE phenotype. In summary, the Adamtsl4(tvrm267) model provides a valuable tool to further elucidate the molecular basis of zonule formation, the pathophysiology of EL and ADAMTSL4 function in the maintenance of the RPE.

Anti-ADAMTS5 monoclonal antibodies: implications for aggrecanase inhibition in osteoarthritis.

The extracellular matrix of articular cartilage is structurally specialized for efficient absorption of mechanical impact. In particular, giant aggregates of the large chondroitin sulfate proteoglycan, aggrecan, with the glycosaminoglycan, hyaluronan, allow cartilage to resist compressive load. Proteolysis of aggrecan by members of the proteinase family ADAMTS (A disintegrin-like and metalloproteinase domain with thrombospondin type 1 motif), was identified as an early step in the inexorable destruction of cartilage in osteoarthritis (OA). Of the investigated proteinases, ADAMTS5 has emerged as a principal mediator of aggrecan loss in OA, convincingly so in mouse models, and with high probability in humans. ADAMTS5 has a bipartite organization, comprising a proteinase domain and an ancillary domain containing exosites for interaction with aggrecan and other substrates. In a recent issue of this journal, Santamaria et al. characterized anti-ADAMTS5 monoclonal antibodies isolated from a phage display library. By blocking the catalytic site of the ADAMTS5 immunogen with a synthetic inhibitor, the authors of the paper biased selection of antibodies to the ancillary domain. This work, together with other antibodies targeting ADAMTS5, offers diverse, high-affinity and, as far as can be determined, selective aggrecanase inhibitors. Mapping of their epitopes provided novel insights into ADAMTS5 interactions with aggrecan. These monoclonal antibodies deserve continued investigation for potential arthritis therapy, although their successful use will require a comprehensive understanding of the physiological roles of ADAMTS5, and its regulation, intrinsic properties and intermolecular interactions.

ADAMTSL2 mutations in geleophysic dysplasia demonstrate a role for ADAMTS-like proteins in TGF-beta bioavailability regulation.

Geleophysic dysplasia is an autosomal recessive disorder characterized by short stature, brachydactyly, thick skin and cardiac valvular anomalies often responsible for an early death. Studying six geleophysic dysplasia families, we first mapped the underlying gene to chromosome 9q34.2 and identified five distinct nonsense and missense mutations in ADAMTSL2 (a disintegrin and metalloproteinase with thrombospondin repeats-like 2), which encodes a secreted glycoprotein of unknown function. Functional studies in HEK293 cells showed that ADAMTSL2 mutations lead to reduced secretion of the mutated proteins, possibly owing to the misfolding of ADAMTSL2. A yeast two-hybrid screen showed that ADAMTSL2 interacts with latent TGF-beta-binding protein 1. In addition, we observed a significant increase in total and active TGF-beta in the culture medium as well as nuclear localization of phosphorylated SMAD2 in fibroblasts from individuals with geleophysic dysplasia. These data suggest that ADAMTSL2 mutations may lead to a dysregulation of TGF-beta signaling and may be the underlying mechanism of geleophysic dysplasia.